M. E. Bisher

Pseudorabies Virus Us9 Directs Axonal Sorting of Viral Capsids

ABSTRACT Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin—a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline—from embryo preparation, embedding, sectioning and staining to visualization—can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.

In Vitro Analysis of Transneuronal Spread of an Alphaherpesvirus Infection in Peripheral Nervous System Neurons

ABSTRACT The neurotropic alphaherpesviruses invade and spread in the nervous system in a directional manner between synaptically connected neurons. Until now, this property has been studied only in living animals and has not been accessible to in vitro analysis. In this study, we describe an in vitro system in which cultured peripheral nervous system neurons are separated from their neuron targets by an isolator chamber ring. Using pseudorabies virus (PRV), an alphaherpesvirus capable of transneuronal spread in neural circuits of many animals, we have recapitulated in vitro all known genetic requirements for retrograde and anterograde transneuronal spread as determined previously in vivo. We show that in vitro transneuronal spread requires intact axons and the presence of the viral proteins gE, gI, and Us9. We also show that transneuronal spread is dependent on the viral glycoprotein gB, which is required for membrane fusion, but not on gD, which is required for extracellular spread. We demonstrate ultrastructural differences between anterograde- and retrograde-traveling virions. Finally, we show live imaging of dynamic fluorescent virion components in axons and postsynaptic target neurons.

Imaging cilia in zebrafish.

Research focused on cilia as extremely important cellular organelles has flourished in recent years. A thorough understanding of cilia regulation and function is critical, as disruptions of cilia structure and/or function have been linked to numerous human diseases and disorders. The tropical freshwater zebrafish is an excellent model organism in which to study cilia structure and function. We can readily image cilia and their motility in embryonic structures including Kupffers vesicle during somite stages and the pronephros from 1 day postfertilization onward. Here, we describe how to image cilia by whole-mount immunofluorescence, transverse cryosection/immunohistochemistry, and transmission electron microscopy. We also describe how to obtain videos of cilia motility in living embryos.

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

Background: Hsc70-auxilin rapidly disassembles clathrin coats from synaptic vesicles for function in neurotransmission. Results: Ssa1p-Swa2p cooperatively disassembles yeast clathrin into coat fragments containing multiple triskelia. Conclusion: Single-particle analysis of yeast clathrin coat disassembly leads to the identification of a partial coat intermediate. Significance: Discovery of a partial clathrin coat intermediate may shed light on coordinated vesicle transport events in the cell. The role of clathrin-coated vesicles in receptor-mediated endocytosis is conserved among eukaryotes, and many of the proteins required for clathrin coat assembly and disassembly have orthologs in yeast and mammals. In yeast, dozens of proteins have been identified as regulators of the multistep reaction required for endocytosis, including those that regulate disassembly of the clathrin coat. In mammalian systems, clathrin coat disassembly has been reconstituted using neuronal clathrin baskets mixed with the purified chaperone ATPase 70-kDa heat shock cognate (Hsc70), plus a clathrin-specific co-chaperone, such as the synaptic protein auxilin. Yet, despite previous characterization of the yeast Hsc70 ortholog, Ssa1p, and the auxilin-like ortholog, Swa2p, testing mechanistic models for disassembly of nonneuronal clathrin coats has been limited by the absence of a functional reconstitution assay. Here we use single-particle burst analysis spectroscopy, in combination with fluorescence correlation spectroscopy, to follow the population dynamics of fluorescently tagged yeast clathrin baskets in the presence of purified Ssa1p and Swa2p. An advantage of this combined approach for mechanistic studies is the ability to measure, as a function of time, changes in the number and size of objects from a starting population to the reaction products. Our results indicate that Ssa1p and Swa2p cooperatively disassemble yeast clathrin baskets into fragments larger than the individual triskelia, suggesting that disassembly of clathrin-coated vesicles may proceed through a partially uncoated intermediate.

Efficient Switching and Domain Interlocking Observed in Polyaxial Ferroelectrics

We present transmission electron microscopy observations of domain wall motion in thin freestanding KNbO 3 crystals under applied electric fields. Since there is no substrate, there is no elastic clamping of 90 domains. We observe that curved and tilted 90 domain walls are the most mobile, whereas untilted 90 domain walls are resistant to field-induced motion. We explain this result in terms of two factors. First, the switching pressure on a domain wall ( P 2 m P 1 ) E is determined by the relative electrostatic energies of the neighboring polarizations P 1 and P 2 . Consequently, some 90 domain walls are immobile under certain field directions, leading to domain interlocking. Second, domain walls experiencing a high switching pressure move by a ripple mechanism, and do not move as rigid sheets. The tilted wall region in such a ripple has a polarization charge, and an associated depolarization field, which reduces the local switching barrier. An accumulation of polarization charge can result in a tilted or curved wall, as occurs at the mobile tips of 90 domain needles. Any increase in density of immobile wall configurations with cycle time represents an inherent contribution to fatigue. Uniaxial ferroelectrics, with polarizations parallel to the field, should not experience such domain interlocking.

Maxwellian charge on domain walls

In situ transmission electron microscopy of domain motion in thinned BaTiO3 and KNbO3 shows that curved ferroelectric domains move more readily under low electric fields than do straight domain walls. We show that this relative motility arises because curved domain walls support a Maxwellian displacement charge and therefore experience a direct pressure that is proportional to the electric field E. Conversely, untilted charge-neutral domain walls experience a pressure proportional to E3 due to induced displacement charge, and therefore tend to resist motion at low fields. Any physical process that leads to an increase in density of the immobile charge-free domain walls, relative to the more mobile curved domain walls, could lead to an increase in overall resistance to domain switching, providing an intrinsic contribution to ferroelectric fatigue.

In-Situ TEM Study of Domain Propagation in Ferroelectric Barium Titanate, and Its Role in Fatigue

We have conducted in-situ transmission electron microscopy (TEM) experiments on thinned single crystal barium titanate in order to study the effects of applied electric field, temperature, electron beam irradiation and UV irradiation on domain nucleation and propagation. We observe two basic modes of domain wall motion; (i) a lateral motion which uniformly widens or narrows the total domain width; (ii) a “zipping” motion in which one end of a domain narrows to a point, which then propagates lengthwise, widening (or narrowing) the domain behind it. Both domain creation and destruction can occur by this latter process. When cooling from above Tc, domain growth usually occurs by the “zipping” motion. We believe that both the lateral and “zipping” modes of motion are related. The “zipping” mode tends to occur in the presence of inhomogeneous long-range strain fields, or when trapped charges are present locally. In some instances, the trapped charge is strong enough to show significant image contrast in bright-field. Domain motion, initiated by heat, electric fields or UV irradiation, moves such charges. A model of domain motion is presented which shows how displacement charge can be injected into the ferroelectric, and which may contribute to the fatigue of these materials.